The objective of this proposal is to understand the cellular and molecular mechanisms whereby CFTR is involved in bone formation and how disruption of this protein, as occurs naturally in Cystic Fibrosis, impairs bone formation to produce osteopenia and osteoporosis. As such, this proposal falls into the areas of research recommended by NIAMS Long Range Plan 2006-2009, to "identify the molecular and cellular mechanisms underlying the pathology of bone diseases" (3C-3). Provided the pleiotropic capability of CFTR to conduct bicarbonate, its role in ATP secretion, and its ability to modulate intracellular calcium levels, and the importance of these processes in skeletal homesotasis, we have designed experiments to address whether CFTR mediates these process in osteoblastic cells and the resultant effect on markers of bone formation. In SA1, we will examine the effect of CFTR inhibition (by both pharmacologic and genomic approaches) on the proliferation (measured by BrdU incorporation) and differentiation (qPCR and Western immunoblotting of markers of bone formation, detection of matrix mineralization) of osteoprogenitors, preosteoblasts,and mature osteoblasts. We will next examine whether alterations in proliferation and differentiation in CFTR-inhibited cells is the result of alterations in acid-base balance in osteoblastic cells. In SA2, we will examine the role of CFTR on the regulation of both intracellular and pericellular pH and the resultant changes in proliferation and matrix mineralization. In the studies, we will use the pH-sensitive BCECF to examine the contribution of CFTR on recovery from acid challenge in the presence and absence of CFTR inhibitors. We will next examine the role of CFTR on the regulation of extracellular pH, which is known to be crucial for mineral formation. In SA3, we will examine whether CFTR is required for load-induced ATP release (luciferin-luciferase detection) and transients in intracellular calcium (using the ratiometric fluorophore Fura-2). In SA4, we will examine the effect of CFTR on the maturation and activation of osteoclasts using in vitro bone resorption assays and correlation with markers of osteoclastogenesis. Relevance: Advances in medicine has greatly prolonged the average lifespan of people born with cystic fibrosis. Yet, as the CF population begins to age, we have become aware that another symptom of CF is weakened bones, increases the risk of fractures. These experiments would suggest how CFTR prevents proper formation of bone. It is hoped that, by understanding how CFTR is involved in bone formation, we would provide insight that would help us and others to develop novel therapeutic strategies to ultimately provide insight into musculoskeletal pathologies, including Cystic Fibrosis- and age-related osteoporosis.